PROJECT SUMMARY With more than five times the number of patients on the wait list than will receive a donor organ in the United States, the field of transplantation is facing a serious donor shortage crisis. Overcoming the organ shortage will require integrated strategies, including a particular focus on overcoming ineffective bio-preservation and stabilization protocols. Longer storage durations will provide the infrastructure required to enable global matching programs, eliminate the need to scramble and conduct unplanned surgeries, and reduce unnecessary waste of quality organs. We believe the method for preserving mammalian organs should employ hibernating and freeze-tolerant strategies in nature that are then further augmented using bioengineering principles. Consequently, we seek to develop a protocol for human organ preservation which will achieve high subzero storage temperatures (ranging from -10 to -20 C) in the presence of extracellular ice, and storage durations of weeks to months, using inspiration from in nature. Our approach is unique in organ/tissue preservation literature since we aim to actively initiate ice propagation in the vasculature and extracellular spaces, rather than extreme means of inhibiting ice crystallization as is the current standard. The presence of non-injurious ice will be essential in achieving longer storage durations, while also playing an important role in the scale-up to human livers. While this program targets the banking of human liver, our discoveries and solutions will be translatable to other tissues and organ systems. In Specific Aim 1, we will adapt endothelial cell-coated microvascular networks already developed by our group3 in order to model and develop strategies to overcome challenges associated with ice propagation. Since endothelial cells in the vasculature will be the most vulnerable to ice propagation, SA#1 will be an essential proof of concept of our novel strategy and we already have promising data. In Specific Aim 2, we will engineer an ice nucleating agent which will promote non-injurious propagation of ice in extracellular spaces. Ice nucleating agents are essential for restricting ice formation to extracellular spaces and have been identified as critical strategies for freezing survival. In Specific Aim 3, we will reprogram cells to descend into a state of `suspended animation' with enhanced stress tolerance, as inspired by nature. We will achieve this using both passive temperature effects as well as using pharmacological agents. We will perform in-depth characterization of the molecular impact of our cellular reprogramming efforts. In each specific aim, we scale up rapidly to rat whole liver while also validating in human livers in order to maximize impact.